Subcellular localization of nitric oxide (NO) synthases with effector molecules is an important regulatory mechanism for NO signalling. In the heart, NO inhibits L-type Ca2+ channels but stimulates sarcoplasmic reticulum (SR) Ca2+ release, leading to variable effects on myocardial contractility. Here we show that spatial confinement of specific NO synthase isoforms regulates this process. Endothelial NO synthase (NOS3) localizes to caveolae, where compartmentalization with beta-adrenergic receptors and L-type Ca2+ channels allows NO to inhibit beta-adrenergic-induced inotropy. Neuronal NO synthase (NOS1), however, is targeted to cardiac SR. NO stimulation of SR Ca2+ release via the ryanodine receptor (RyR) in vitro, suggests that NOS1 has an opposite, facilitative effect on contractility. We demonstrate that NOS1-deficient mice have suppressed inotropic response, whereas NOS3-deficient mice have enhanced contractility, owing to corresponding changes in SR Ca2+ release. Both NOS1-/- and NOS3-/- mice develop age-related hypertrophy, although only NOS3-/- mice are hypertensive. NOS1/3-/- double knockout mice have suppressed beta-adrenergic responses and an additive phenotype of marked ventricular remodelling. Thus, NOS1 and NOS3 mediate independent, and in some cases opposite, effects on cardiac structure and function.
A decrease in Ca(SR) is sufficient to explain the diminished Ca(i) transients in F, without a change in the effectiveness of coupling. Therefore, therapeutic approaches that increase Ca(SR) may be able to fully correct the Ca handling deficit in heart failure.
Abstract-Increased Na ϩ -Ca 2ϩ exchange (NCX) activity in heart failure and hypertrophy may compensate for depressed sarcoplasmic reticular Ca 2ϩ uptake, provide inotropic support through reverse-mode Ca 2ϩ entry, and/or deplete intracellular Ca 2ϩ stores. NCX is electrogenic and depends on Na ϩ and Ca 2ϩ transmembrane gradients, making it difficult to predict its effect on the action potential (AP). Here, we examine the effect of [Na ϩ ] i on the AP in myocytes from normal and pacing-induced failing canine hearts and estimate the direction of the NCX driving force using simultaneously recorded APs and Ca 2ϩ transients. AP duration shortened with increasing [Na ϩ ] i and was correlated with a shift in the reversal point of the NCX driving force. At [Na ϩ ] i Ն10 mmol/L, outward NCX current during the plateau facilitated repolarization, whereas at 5 mmol/L [Na ϩ ] i , NCX had a depolarizing effect, confirmed by partially inhibiting NCX with exchange inhibitory peptide. Exchange inhibitory peptide shortened the AP duration at 5 mmol/L [Na ϩ ] i and prolonged it at [Na ϩ ] i Ն10 mmol/L. With K ϩ currents blocked, total membrane current was outward during the late plateau of an AP clamp at 10 mmol/L [Na ϩ ] i and became inward close to the predicted reversal point for the NCX driving force. The results were reproduced using a computer model. These results indicate that NCX plays an important role in shaping the AP of the canine myocyte, helping it to repolarize at high [Na ϩ ] i , especially in the failing heart, but contributing a depolarizing, potentially arrhythmogenic, influence at low [Na Key Words: heart failure Ⅲ Na ϩ -Ca 2ϩ exchanger Ⅲ reversal potential Ⅲ Ca 2ϩ transients T he Na ϩ -Ca 2ϩ exchanger (NCX) catalyzes the electrogenic exchange of Na ϩ for Ca 2ϩ across the cardiac sarcolemma and is reversible, operating in either forward (Ca 2ϩ -efflux) or reverse (Ca 2ϩ -influx) modes, depending on the prevailing electrochemical driving forces for Ca 2ϩ and Na ϩ . This complex dependence on transmembrane ion and voltage gradients, which are both rapidly changing during the cellular action potential (AP), makes predictions about the overall influence of NCX current (I NCX ) on excitation-contraction coupling challenging. NCX is the primary Ca 2ϩ extrusion mechanism in the heart 1,2 and is required to remove the increment of Ca 2ϩ entering the myocyte via Ca 2ϩ channels on each beat, 3 but the timing of the transition from reverse-mode exchange (outward I NCX ) to forward-mode exchange (inward I NCX ) has been difficult to determine. Because the membrane impedance during the AP plateau of large mammals (including humans) is high, the net direction of current flow through NCX is likely to be a fundamental determinant of AP duration (APD).Early investigations into the net effect of I NCX on the AP demonstrated that it contributed net inward current and prolonged the AP in the rat. 4 than that reported by intracellular Ca 2ϩ dyes. In the failing heart, where SR function is impaired, a greater dependence on NCX for...
Defective excitation-contraction coupling in heart failure is generally associated with both a reduction in sarcoplasmic reticulum (SR) Ca(2+) uptake and a greater dependence on transsarcolemmal Na(+)-Ca(2+) exchange (NCX) for Ca(2+) removal. Although a relative increase in NCX is expected when SR function is impaired, few and contradictory studies have addressed whether there is an absolute increase in NCX activity. The present study examines in detail NCX density and function in left ventricular midmyocardial myocytes isolated from normal or tachycardic pacing-induced failing canine hearts. No change of NCX current density was evident in myocytes from failing hearts when intracellular Ca(2+) ([Ca(2+)](i)) was buffered to 200 nmol/L. However, when [Ca(2+)](i) was minimally buffered with 50 micromol/L indo-1, Ca(2+) extrusion via NCX during caffeine application was doubled in failing versus normal cells. In other voltage-clamp experiments in which SR uptake was blocked with thapsigargin, both reverse-mode and forward-mode NCX currents and Ca(2+) transport were increased >2-fold in failing cells. These results suggest that, in addition to a relative increase in NCX function as a consequence of defective SR Ca(2+) uptake, there is an absolute increase in NCX function that depends on [Ca(2+)](i) in the failing heart.
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